Fig. 5. cAMP pathway analysis and in vitro and in vivo
validation. (A) cAMP signaling pathway remodeling. Significant (two-tailed t test, P < 0.05) transcript level
decreases (blue) and increases (red) in all macrophage
states relative to Mo, as well as b-glucan treatment-specific increases,
are indicated; see also fig. S8. (B) Diagram of the timeline and experimental setup of the in vitro cAMP inhibition experiment. cAMP inhibitors
or vehicle were applied to human primary monocytes during the first 24 hours
of “training” [see fig. S4E and (70) for details]. After 6 days, secondary
stimulation of cells was performed with LPS to induce cytokine production.
(C to E) The inhibitor of adenylate cyclase 2′,5′-dideoxyadenosine (ddA), the
PKA inhibitor H89, and the b-adrenergic receptor blocker propranolol
inhibited the induction of training by b-glucan, assayed as the response to
LPS stimulus on day 6. *P < 0.05 (Wilcoxon signed-rank test). Data show the
fold increase in cytokine production (ELISA) upon training as compared with
untrained Mf cells and are presented as mean T SEM, n > 6 in three inde-
pendent experiments. (F) Timeline of the in vivo training experiment in mice.
Mice were either injected intravenously with phosphate-buffered saline (con-
trol) or a nonlethal dose of C. albicans [2 × 104 colony-forming units (CFU)
per mouse] 7 days before intravenous inoculation of a lethal C. albicans
dose (2 × 106 CFU per mouse). In a third group of mice, propranolol was
administered before the inoculation of the nonlethal dose of C. albicans
(70). (G) Survival rate of wild-type C57BL/6 mice to a systemic infection
with C. albicans (n ≥ 8 per group). Statistical significance between the groups
(Kaplan and Meier) was calculated using the log-rank test. *P < 0.05, **P <
0.01 versus control animals.